Project #3 Goals and Objectives: The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines are important for many systems related to growth and differentiation. Our studies are concerned with how these polyamines are synthesized and metabolized and their physiologic functions, how they act in vivo. In this project we are particularly interested in elucidating the functions of polyamine conjugated glutathione (glutathionylspermidine) in E. coli by the use of microarrays, proteomics and bioinformatics techniques. Summary: We continued to be interested in the physiologic function of glutathionylspermidine. We have carried out taxonomic analysis of the sequences that encode glutathionlyspermidine synthase/amidase, and have found that homologous sequences are almost completely restricted to Enterobacter and several members of the Kinetoplastida phyla (including trypanosomes), and are strikingly absent in all invertebrate and vertebrate species, Archea, plants and most Eubacteria (with only a few exceptions). Particularly striking is the very high homology (>60%) among different Enterobacterial species. Although highest accumulation of glutathionylspermidine is found in stationary phase cultures where most of the intracellular spermidine is converted to glutathionylspermidine, our isotope exchange experiments show that there is a rapid exchange between intracellular spermidine and glutathionylspermidine even in log phase cells. Our earlier microarray studies comparing gss+ and gss- strains of E. coli show that a large number of genes are either upregulated or downregulated by the loss of the gss gene. Most significant categories of up-regulated genes include sulfur utilization, glutamine and succinate metabolism, polyamine and arginine metabolism, and purine and pyrimidine metabolism. Subsequently we performed proteomic studies (in collaboration with Dr. Eric Anderson of the NIDDK Mass Spectrometry section) comparing the protein composition of the two strains using the SILAC technique. For this purpose we have constructed strains containing deletions in the lysA and argA genes in the gss+ and gss&#916; background. From the SILAC assay we found that 12 proteins were up-regulated and 8 proteins were down-regulated in gss- cells as compared to gss+ cells. Some of the up-regulated proteins are ZapA, YghU, MgtA, AroH, Ecfk, Pal. The down-regulated peptides include Flu, GlpX, FldA, YcbB, DscB, YghD. Most striking was the change in the Flu protein. However, on further investigation, we found that the change in the Flu protein was not related to the gss deletion, but rather was the result of the matastable nature of the flu gene. This is a very interesting protein and is the phase-variable outer membrane protein Ag43. On further studies we found that the background strain used as the parent for the construction of the gss&#916; had a particularly high frequency of conversion of flu+ to flu-. For further studies we are isolating gss+/gss&#916; strains with identical expression of flu proteins, which will be used for SILAC and other comparative analyses to find out the physiological significance of glutathionylspermidine in E. coli.